Nitric oxide is known to modulate cardiac contractility, however the subcellular mechanisms are unknown. There are three major regulators of cardiac contractility: L-type calcium channel (ICa), sarcoplasmic reticulum (SR), and the contractile proteins. Work has been done on the effects of NO on the ICa and the contractile proteins; however, little work has been done on the effects of NO on the SR (especially in intact myocytes). The long term objective is to understand the role of NO in cardiac excitation-contraction coupling, and thus its role in modulating cardiac contractility. The specific aims of this proposal are: 1) to determine if the anti-adrenergic effect of NO can be explained by inhibition of ICa in isolated beta-adrenergic stimulated cardiac myocytes, 2) to determine the effects of NO on specific SR calcium handling processes after beta-adrenergic stimulation (calcium handling by the ryanodine receptor (RyR), SR calcium content, and calcium uptake via the Ca-ATPase) and 3) to determine subcellular signaling mechanisms of NO mechanisms of NO regulation of SR function in isolated beta-adrenergic stimulated cardiac myocytes. The hypothesis is that NO will have an inhibitory effect on SR function (negative inotropic agent), and this effect is due to cGMP-dependent and cGMP-independent signaling pathways.